SUMMARY OF WORK The chemotaxis, or directed migration, of vascular smooth muscle cells (VSMCs) is critical to the lesion formation of atherosclerosis and post-angioplasty restenosis. While the directional cues for guiding this movement are shown to come from locally-released chemoattractants like platelet-derived growth factor (PDGF), the regulatory cytosolic signals have yet to be defined. The ubiquitous signal transduction pathway of cytosolic calcium (Cai) has been suggested as a viable mediator. Therefore, we examined the regulatory role of Cai in VSMC chemotaxis. There are two distinct, sequential Cai signaling phases in response to a PDGF gradient: (1) an initial phase (time frame of minutes), with a relatively synchronous, transient increase in Cai in most cells, and (2) a delayed phase (time frame of hours), with asynchronous increases in Cai among individual cells which precede chemotaxis. All cells prior to undergoing chemotaxis displayed late, marked elevations in Cai, while most cells that failed to undergo chemotaxis exhibited little to no change in Cai. There appears to be a Cai threshold (400 nM) for chemotaxis, since all migrating cells achieved this level, and chemotaxis was inhibited in cells prevented from achieving at least this level by buffering the delayed (but not the initial) Cai rise with the Cai -chelator BAPTA. Neither chemotaxis nor late increases in Cai were observed in VSMCs in the absence of PDGF or of its gradient. While Ca2+ release from internal stores (and Ca2+ oscillations) are not necessary for chemotaxis, trans-sarcolemmal Ca2+ influx does provide a principal source for the delayed-phase Cai signaling. Putative "receptor-operated" Ca2+ channels (and not L-type, T-type, or mechano- sensitive Ca2+ channels) are contributing sites for this Ca2+ influx. In summary, the Cai mobilization that is uniquely mediated by a PDGF gradient does have a regulatory capacity in the VSMC chemotactic response. The achievement of an apparent Cai threshold may serve, in part, to initiate the Ca2+-dependent activation of CaMK II, a process critical to chemotaxis.